1. Field of the Invention
This invention pertains to the field of inhibition of angiogenesis by delivery of angiogenesis inhibitors derived from plasminogen. The angiogenesis inhibitors are delivered in polypeptide or nucleic acid form.
2. Background
Angiogenesis, the process by which new blood vessels are formed, is essential for embryonic development and other normal physiological processes such as wound healing and formation of the corpus luteum, endometrium and placenta. However, when angiogenesis occurs at an inappropriate time or location, numerous disease states and other undesirable conditions sometimes arise. For example, angiogenesis is involved in other diseases and conditions, including arthritis and atherosclerotic plaques, diabetic retinopathy, neovascular glaucoma, trachoma and corneal graft neovascularization, psoriasis, scleroderma, hemangioma and hypertrophic scarring, vascular adhesions and angiofibroma.
Angiogenesis is also essential for solid tumor growth and metastasis. Folkman (1990) J. Nat""l. Cancer Inst. 82: 4-6; Kim et al. (1993) Nature 362: 841-844; Hori et al. (1991) Cancer Res. 51: 6180-6184; Millauer et al. (1994) Nature 367: 576-579; Sim et al. (1997) Cancer Res. 57: 1329-1334. Tumor cells are believed to cause a local disruption of the delicate balance that normally exists between angiogenesis inhibitors and stimulators. According to this model, by producing angiogenesis stimulators, tumors cause a local increase in the ratio of stimulators to inhibitors, which induce the formation of new blood vessels that carry oxygen and nutrients to the growing tumor. These factors include vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF), basic and acidic fibroblast growth factors, interleukin-1, hepatocyte growth/scatter factor (HGF) and others. See, e.g., O""Reilly (1997) Regulation of Angiogenesis, Goldberg and Rosen, Eds., Birkhauser Verlag, Basel, pp. 273-294.
Angiostatin, which is an angiogenesis inhibitor, is a naturally-occurring internal cleavage product of plasminogen. Angiostatin has been estimated to have a molecular weight of between approximately 38 kilodaltons and 45 kilodaltons as determined by reducing polyacrylamide gel electrophoresis. However, the precise boundaries of the angiostatin fragment of plasminogen have not been definitively identified. The angiostatin fragment of plasminogen was reported to begin at about amino acid 98 of a murine plasminogen polypeptide, and at either amino acid 97 or 99 of an intact human plasminogen polypeptide.
Human plasminogen has five characteristic protein domains called xe2x80x9ckringle structures.xe2x80x9d Murine plasminogen has four or five kringle structures; it is unclear whether the amino acid sequence of murine plasminogen corresponding to the second kringle structure of murine plasminogen forms a kringle structure as is present in human plasminogen. Based on the estimated molecular weight, human angiostatin was predicted to include kringles 1-3 and a part of kringle 4 of the five plasminogen kringle region (see, e.g., Robbins, K. C. (1987) Hemostasis and Thrombosis, Basic Principles and Practice, 2nd Edition, ed. Colman, R. W. et al., J. B. Lippincott Company, pp. 340-357). Each kringle region of the plasminogen molecule has approximately eighty amino acids and three disulfide bonds. The complete sequence of plasminogen contributes the signal peptide for secretion and contains sites for protease cleavage to liberate angiostatin once plasminogen is secreted.
Recent research indicates tumor growth is dependent on angiogenesis and that inhibiting angiogenesis can slow tumor growth. Kim et al. (1993) Nature 362: 841-844; Weidner et al. (1991) New Engl. J. Med. 324: 1-7. Therefore, a need exists for compounds and methods for inhibiting undesirable angiogenesis. Such methods and compounds will find use not only in cancer therapy, but also for treating other angiogenesis-associated conditions. The present invention fulfills these and other needs.
The present invention provides methods of delivering anti-angiogenic activity to a mammal. In one embodiment, the method involves introducing into cells of the mammal a nucleic acid that includes a polynucleotide sequence encoding a polypeptide which comprises an amino acid sequence substantially identical to at least three kringle domains of native plasminogen. The anti-angiogenic polypeptides generally have an amino acid sequence extending from about amino acid 97 to at least about amino acid 462 of plasminogen.
Also provided by the invention are methods of inhibiting angiogenesis in a mammal by administering to the mammal a nucleic acid comprising a polynucleotide sequence encoding an anti-angiogenic polypeptide which comprises at least three kringle domains of plasminogen. The sequence encoding the anti-angiogenic polypeptide generally is operably linked to a polynucleotide sequence encoding a signal peptide.
The invention also provides methods of treating a condition that is associated with undesirable endothelial cell proliferation. These methods involve administering to the mammal a nucleic acid comprising a polynucleotide sequence encoding an anti-angiogenic polypeptide which comprises at least three kringle domains of plasminogen. The sequence encoding the anti-angiogenic polypeptide is operably linked to a polynucleotide sequence encoding a signal peptide.
In another embodiment, the invention provides an isolated nucleic acid that comprises an expression cassette that includes a polynucleotide sequence encoding a signal peptide operably linked to a polynucleotide sequence encoding an anti-angiogenic polypeptide. The anti-angiogenic polypeptide includes at least kringles 1-3 of plasminogen.
Also provided by the invention are isolated anti-angiogenic polypeptides which include at least three kringle regions of plasminogen. In a preferred embodiment, the polypeptides include kringles 1-4 of human plasminogen and are less than full length plasminogen.
The invention also provides endothelial cells and tumor cells that contain a recombinant expression cassette which includes a polynucleotide sequence encoding a signal peptide operably linked to a polynucleotide sequence encoding an anti-angiogenic polypeptide. The anti-angiogenic polypeptides include at least three kringles of plasminogen, preferably kringles 1-4.